This invention relates generally to the field of recombinant retroviral particles for use in gene delivery and more specifically to packaging cell lines for producing retroviral particles.
Retroviruses are enveloped RNA viruses that, after infection of a host cell, reverse transcribe their RNA genomes into a DNA intermediate, or provirus. All viruses containing an RNA genome and producing an RNA-dependent DNA polymerase are contained in the retroviral family. The family is divided into three subfamilies: (1) Oncovirinae, including all the oncogenic retroviruses, and several closely related non-oncogenic viruses; (2) Lentivirinae, the xe2x80x9cslow retrovirusesxe2x80x9d such as the human immunodeficiency virus (HIV) and visna virus; and (3) Spumavirinae, the xe2x80x9cfoamyxe2x80x9d retroviruses that induce persistent infections, generally without causing any clinical disease. Retroviruses containing at least three types of proteins encoded by the viral genome, i.e., gag proteins (the group antigen internal structural proteins), pol proteins (the RNA-dependent DNA polymerase and the protease and integrase proteins), and env proteins (the viral envelope protein or proteins). In addition to genes encoding the gag, pol, and env proteins, the genome to the retrovirus includes two long terminal repeat (LTR) sequences, one at the 5xe2x80x2 and one at the 3xe2x80x2 end of the virus. These 5xe2x80x2 and 3xe2x80x2 LTRs promote transcription and polyadenylation of viral mRNAs and participate in the integration of the viral genome into the cellular DNA of the host.
The provirus can be stably integrated into the host""s cellular DNA. Gene products encoded by the provirus are then expressed by the host cell to produce retroviral virions, thereby replicating the virus. Because the retroviral genome can be manipulated to include exogenous nucleotide sequence(s) of interest for expression in a target cell, retroviral vectors are important tools for stable gene transfer into mammalian cells.
Many proposed gene therapy applications use retroviral vectors to take advantage of the ability of these naturally infectious agents to transfer and efficiently express recombinant nucleotide sequences in susceptible target cells. Retroviral vectors suitable for use in such applications are generally defective retroviral vector that are capable of infecting the target cell, reverse transcribing their RNA genomes, and integrating the reverse transcribed DNA into the target cell genome, but are incapable of replicating within the target cell to produce infectious retroviral particles (e.g., the retroviral genome transferred into the target cell is defective in gag, and/or in pol, see Coffin, J., In: RNA Tumor Viruses, Weiss, R. et al., (ed) Cold Spring Harbor Laboratory, Vol. 2, pp. 36-73, 1985).
Retroviral vectors and packaging cells (helper cells) have been developed to introduce recombinant nucleic acid molecules into mammalian cells without the danger of the production of replicating infectious virus. This methodology uses two components, a retroviral vector and a packaging cell. The retroviral vector contains long terminal repeats (LTRs), the foreign DNA to be transferred, and a packaging sequence. This retroviral vector will not reproduce by itself because the genes which encode the structural and envelope proteins are not included within the vector. The packaging cell contains genes encoding the gag, pol, and env proteins, but does not contain the packaging signal, so that the cell can only form empty virus particles by itself. With this method, the retroviral vector is introduced into the packaging cell, to create a cell able to produce virus. The cell manufactures viral particles containing only the retroviral vector DNA, and therefore has been considered safe.
Use of retroviral vectors is limited in many aspects. For example, although retroviruses can efficiently infect and stably integrate into the genome of rapidly-dividing cells, retroviral integration into the genome of non-dividing or slowly dividing cells is inefficient (Springett et al. 1989 J. Virol. 63:3865-3869; Miller et al. 1990 Mol. Cell. Biol. 10:4239-4242; Roe et al. 1993 EMBO J. 12:2099-2108). Most packaging systems provide only modest vector titers, and the fragility of retroviral vector particles complicate purification and concentration (Paul et al. 1993 Hum. Gene Therap. 4:609-615). Finally, retroviruses enter target cells by binding of retroviral envelope glycoproteins (encoded by the env gene) to specific target cell surface receptors. This envelope protein-cell surface receptor interaction is often species specific, and in some cases even tissue specific. Moreover, the level of expression of the cell surface receptor on the target cells can vary widely among target cells. As a result, retroviruses usually have a limited host range (Kavanaugh et al. 1994 Proc. Natl. Acad. Sci. USA 91:7071-7075; Hopkins 1993 Proc. Natl. Acad. Sci. USA 90:8759-8760).
One strategy for both expanding retroviral host cell range and structural stability of the retroviral virion involves production viral vectors. Pseudotyped retroviral vectors useful in transformation of target cells are generally composed of retroviral virion structural proteins (e.g., gag proteins), a recombinant RNA genome containing the nucleotide sequence of interest, the pol protein for reverse transcription of the recombinant RNA contained in the virion, and a non-retroviral envelope protein or an envelope protein from a different retrovirus. The recombinant RNA genome is usually replication defective, e.g., defective in the pol and/or gag genes, to prevent production of infectious retrovirus following transfer of the nucleotide sequence of interest into the target cell. The envelope protein of the pseudotyped retroviruses normally selected to provide a broader host range or to provide selective targeting of cells to be infected.
The present invention provides packaging cell lines and recombinant retroviral particles produced therefrom, particularly pseudotyped retroviral particles. Exemplary packaging cell lines are derived from 293 HeLa, Cf2Th, D17, MDCK, or BHK cells, most preferably from 293 cells. Retroviral particles are produced by inducibly expressing an envelope protein of interest (e.g., a retroviral envelope or the envelope protein of vesicular stomatitis virus (VSV G)). Inducible expression of the envelope protein is accomplished by operably linking an envelope protein-encoding nucleotide sequence to an inducible promoter (e.g., a promoter composed of a minimal promoter linked to at least one copy of tetO, the binding site for the tetracycline repressor (tetR) of the Escherichia coli tetracycline resistance operon Tn10). Expression from the inducible promoter is regulated by a transactivating factor, composed of a first ligand-binding domain that negatively regulates transcription from the inducible promoter (e.g., a prokaryotic tetracycline repressor polypeptide (tet R)). Transcription of the envelope-encoding nucleotide sequence under control of the inducible promoter is activated by a transactivator when tetracycline is absent.
A primary object of the invention is to provide a packaging cell line that produces retroviral particles, especially pseudotyped retroviral particles, more efficiently (e.g., at higher titers) than conventional packaging cell lines. The packaging cell line of the invention is characterized by a first polynucleotide having an HIV genome operably linked to a first inducible promoter wherein the HIV genome is defective for cis-acting elements, for self-replication and for expression of functional Env protein; a second polynucleotide encoding a functional heterologous Env protein operably linked to a second inducible promoter; and a third polynucleotide encoding a regulatable transcriptional activator controlling transcription from the first and second inducible promoters.
Another object of the invention is to provide recombinant retroviral vectors, especially pseudotyped retroviral vectors, that are useful in both in vitro and in vivo transformation of a target cell.
An advantage of the invention is that packaging cell lines can be produced that contain an envelope-encoding nucleotide sequence stably integrated in the cell""s genome, which sequence can be inducibly expressed, thus allowing generation of packaging cell lines capable of expressing an envelope protein that is otherwise toxic to the host cell (e.g., VSV G). Another advantage of the invention is that the packaging cell lines do not have the potential to produce replication competent retroviruses.
In another embodiment, the invention provides a method for producing a packaging cell line. The method includes transfecting a suitable cell with i) a first polynucleotide having an HIV genome operably linked to a first inducible promoter wherein the HIV genome is defective for cis-acting elements, for self-replication and for expression of functional Env protein; ii) a second polynucleotide encoding a functional heterologous Env protein operably linked to a second inducible promoter; and iii) a third polynucleotide encoding a regulatable transcriptional activator controlling transcription from the first and second inducible promoters; and culturing the transfected cells above under conditions which allow screening for cells containing proteins encoded by the polynucleotides. Preferably, the cell is co-transfected with a selectable marker encoding polynucleotide, such as an antibiotic resistance gene.
In another embodiment, the invention provides a method of detecting a viral nucleic acid sequence integrated into a target nucleic acid sequence by reversibly immobilizing the target nucleic acid sequence to a solid support, contacting the target nucleic acid with a viral preintegration complex (PIC) containing the viral nucleic acid sequence, under such conditions and for sufficient time so as to allow the viral nucleic acid sequence to integrate into the target nucleic acid sequence and detecting the integrated viral nucleic acid sequence.
In yet another embodiment, the invention provides a method of detecting viral integrase activity contained in viral preintegration complex (PIC). The method includes reversibly immobilizing a target nucleic acid sequence to a solid support; contacting the target nucleic acid with the viral preintegration complex (PIC) containing a viral nucleic acid sequence, under such conditions and for sufficient time so as to allow the viral nucleic acid sequence to integrate into the target nucleic acid sequence and detecting the integrated viral nucleic acid sequence. The presence of the viral nucleic acid sequence in the target nucleic acid sequence is indicative of integrase activity.
In a further embodiment, the invention provides a method of identifying a compound that affects viral integrase activity contained in a preintegration complex (PIC). The method includes reversibly immobilizing a target nucleic acid sequence to a solid support; contacting the target nucleic acid with the viral preintegration complex (PIC) containing a viral nucleic acid sequence and a test compound suspected of affecting integrase activity, under such conditions and for sufficient time so as to allow the viral nucleic acid sequence to integrate into the target nucleic acid sequence; and detecting the integrated viral nucleic acid sequence. The amount of viral nucleic acid sequence integrated in the target nucleic acid sequence is indicative of the effect of the compound on integrase activity.
These and other objects, advantages and features of the present invention will become apparent to those persons skilled in the art upon reading the details of the vectors, cell lines and methodology as more fully set forth below.